The field of the present invention is the area of cellulolytic enzymes, nucleotide sequences encoding them and recombinant host cells and methods for producing them.
Cellulosic biomass, photosynthesized by solar energy with CO.sub.2 and H.sub.2 O, is one of the most important renewable energy resources on earth. Its effective utilization through biological processes is one approach to overcoming the shortage of foods, feeds and fuels, expected as a consequence of the explosive increase in human population [Ohmiya et al. (1997)]. Several types of enzymes are required for complete hydrolysis of cellulose to glucose, including endoglucanase, exoglucanase or cellobiohydrolase and .beta.-glucosidase [Filho 1996)].
Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous animals. Since the first anaerobic fungus, Neocallimastix frontalis was isolated in 1975 from the rumen of sheep [Orpin, G. C. (1975) J. Gen. Microbiol. 91, 249-262], at least 17 different anaerobic fungi have been isolated from ruminant and nonruminant herbivores. Anaerobic fungi produce highly active hydrolytic enzymes [Borneman et al.(1989) Appl. Environ. Microbiol. 55, 1066-1073], they physically associate with the lignocellulosic tissue of plant fragments, and their hyphae penetrate the plant tissue in vivo [Akin et al.(1983) Appl. Environ. Microbiol. 46, 738-748; Thedorou et al. (1996) Proc. Nutr. Soc. 55, 913-926]. These fungi are involved in degradation of plant biomass and play an important role in the rumen ecosystem. Several genes coding for hydrolytic enzymes have been cloned and sequenced from the monocentric fungi Neocallimastix patriciarum [Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072; Zhou et al. (1994) Biochem. J.. 297, 359-364; Black et al. (1994) Biochem. J.. 299, 381-387; Denman et al. (1996) Appl. Environ. Microbiol. 62, 1889-1896; Dalrymple et al. (1997) Microbiology 143, 2605-2614], N. frontalis [Reymond et al. (1991) FEMS Microbiol. Letts. 77, 107-112; Durand et al. (1996) Curr. Genet. 30, 531-540], and Piromyces sp. [Fanutti et al. (1995) J. Biol. Chem. 270, 29314-29322] and from the polycentric fungi Orpinomyces PC-2 [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635; Chen et al. (1997) J. Bacteriol. 179, 6028-6034] and Orpinomycesjoyonii [Liu et al. (1997) Can. J. Microbiol. 43, 477-485]. It has been suggested that genes encoding three mannanases of a Piromyces sp. [Millward-Sadler et al. (1996) FEMS Microbiol. Letts. 141, 183-188] and two cellobiohydrolases (CelA and CeiC) of Orpinomyces PC-2 [Li et al. (1997) Appl. Environ. Microbiol. 63, 4721-4728; see also WO 98/14597] resulted from gene duplications. CelB is described in WO 98/14597, incorporated by reference herein in its entirety.
There is a need in the art for a high-specific-activity cellulase in pure form which degrades cellulosic materials, and for DNA encoding this cellulase to enable methods of producing the cellulase in pure form.